WO2021217597A1 - 一种具有高扩增活性的耐热dna聚合酶突变体 - Google Patents

一种具有高扩增活性的耐热dna聚合酶突变体 Download PDF

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WO2021217597A1
WO2021217597A1 PCT/CN2020/088341 CN2020088341W WO2021217597A1 WO 2021217597 A1 WO2021217597 A1 WO 2021217597A1 CN 2020088341 W CN2020088341 W CN 2020088341W WO 2021217597 A1 WO2021217597 A1 WO 2021217597A1
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dna polymerase
mutant
taq enzyme
pcr
seq
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PCT/CN2020/088341
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English (en)
French (fr)
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蒋析文
刘霭珊
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广州达安基因股份有限公司
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Priority to CN202080100330.1A priority Critical patent/CN115485374A/zh
Priority to PCT/CN2020/088341 priority patent/WO2021217597A1/zh
Priority to EP20761492.6A priority patent/EP3922718A4/en
Priority to US16/957,276 priority patent/US20220325259A1/en
Publication of WO2021217597A1 publication Critical patent/WO2021217597A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the invention belongs to the field of biotechnology. Specifically, the invention relates to a heat-resistant DNA polymerase mutant with high amplification activity.
  • Taq enzyme is a heat-resistant DNA polymerase derived from the heat-resistant bacterium Thermus aquaticus, with a molecular weight of 94KDa. In the presence of magnesium ions, its optimal reaction temperature is 75-80°C, and its active half-life at 95°C It is 40 minutes and has 5'-3' exonuclease activity. Because of its high temperature resistance, it is widely used in polymerase chain reaction (PCR) and is the first choice for nucleic acid amplification and detection reactions.
  • PCR polymerase chain reaction
  • Commercial Taq enzyme is cloned and expressed using E. coli prokaryotic expression system.
  • Taq enzyme There are several ways to modify Taq enzyme. 1: Increase the structural domain to give it new properties. For example, adding single-strand binding domain (SSB) or DNA binding protein Sso7 can enhance the binding ability of Taq enzyme to primer and template DNA, making it have stronger extension ability and continuous synthesis ability, suitable for the amplification of long fragment DNA reaction. However, increasing the domain will directly increase the molecular weight of Taq enzyme, which may reduce the solubility and stability of Taq enzyme. Reduce the yield of prokaryotic expression production. 2: Remove unnecessary domains on Taq enzyme.
  • SSB single-strand binding domain
  • Sso7 DNA binding protein binding protein Sso7
  • the Taq enzyme mutant obtained by this method does not have 5'-3' exonuclease activity, and is not suitable for quantitative PCR reactions based on the Taqman probe method, and its useful range is limited. 3: Site-directed mutation method.
  • Site-directed mutagenesis of amino acids in active sites, magnesium ion binding sites, and DNA binding sites improves the affinity of each site to substrates, templates and primers, thereby improving tolerance to various inhibitors. Due to the complexity of the protein structure, some amino acids far away from the active site may also affect the overall structure of the enzyme. Therefore, it is difficult to modify the enzyme as a whole by only mutating the amino acids at a few specific active center sites. Moreover, the existing computer simulation technology is difficult to predict what effect each site mutation will have on the overall structure. The use of site-directed mutagenesis to prepare mutants and carry out mutant screening is very heavy and inefficient, and some may have a significant impact on activity. The site could not be recognized.
  • the object of the present invention is to provide a heat-resistant DNA polymerase mutant with high amplification activity.
  • a mutated DNA polymerase which is mutated at one or more sites selected from the group consisting of: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, H785, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.2.
  • the activity of the mutant DNA polymerase is at least 1.5 times that of the wild-type DNA polymerase (SEQ ID NO.: 2); preferably at least 2 times; more preferably at least 3 times.
  • amino acid sequence of the wild-type DNA polymerase is shown in SEQ ID NO.: 2.
  • the amino selection sequence of the mutant DNA polymerase has at least 80% homology compared with SEQ ID NO. 2; more preferably, has at least 90% homology; most preferably It has at least 95% homology; such as at least 96%, 97%, 98%, 99% homology.
  • mutant DNA polymerase is selected from the group of mutants 1-20:
  • the number of mutation sites in the mutated DNA polymerase is 1-4, preferably 2 or 3.
  • the mutant DNA polymerase is selected from each specific mutant enzyme in Table 2.
  • the mutant DNA polymerase includes the mutation sites of each specific mutant enzyme in Table 2.
  • the mutated DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.: 2, and the mutated DNA polymerase includes a mutation site selected from the following group point:
  • the second aspect of the present invention provides a polynucleotide molecule encoding the mutant DNA polymerase of the first aspect of the present invention.
  • the third aspect of the present invention provides a vector containing the nucleic acid molecule according to the second aspect of the present invention.
  • the fourth aspect of the present invention provides a host cell containing the vector according to the first aspect of the present invention or the chromosome integrated with the nucleic acid molecule according to the second aspect of the present invention.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is Escherichia coli.
  • the eukaryotic cell is a yeast cell.
  • the fifth aspect of the present invention provides a method for preparing the mutant DNA polymerase of the first aspect of the present invention, including the steps:
  • the temperature for culturing the host cell in the step (i) is 20°C-40°C; preferably 25°C-37°C, such as 35°C.
  • the sixth aspect of the present invention provides a kit comprising the mutant DNA polymerase described in the first aspect of the present invention.
  • the present inventors applied protein directed evolution technology to construct a random mutation library for the polymerase active domain of Taq enzyme. By gradually adding screening pressure, unsuitable mutations were naturally eliminated and mutations with dominant traits were eliminated. Gradually accumulate, and finally screen out a series of amino acid sites and their mutations that play a key role in the amplification and polymerization performance of Taq enzyme, and obtain Taq enzyme mutants with high amplification performance. On this basis, the present invention has been completed.
  • Taq enzyme is widely used in polymerase chain reaction (PCR) and is the first choice for nucleic acid amplification and detection reactions.
  • PCR polymerase chain reaction
  • Commercial Taq enzyme is cloned and expressed using E. coli prokaryotic expression system.
  • the DNA sequence of the wild-type Taq enzyme is as follows:
  • amino acid sequence of the wild-type Taq enzyme is as follows:
  • the present invention screens out amino acid sites highly related to Taq enzyme amplification activity and their mutation modes by means of directed evolution.
  • Related mutant amino acid sites include: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, and H785.
  • the number of amino acid residues is based on SEQ ID NO.: 2. Mutation of the above amino acid position into any other amino acid can obtain a taq enzyme mutant with higher activity.
  • the preferred mutation forms include: E507A/Q/H/M, K508L, E734G/F/M, F749K/G/V /T/E, L764K/Q, V453A/G, R737K/W/P, T757S/W, H785G/L/K, S624T/K, Y672R/P, A518Q, F495G/R, T509L.
  • the present invention is by directed evolution, selected amino acid sites, and mutants manner highly relevant activity of Taq mutant library from a random mutation is 105 times the amount thereof directed mutagenesis, screening is more conducive to a synergistic effect Mutation sites, and these sites cannot be predicted by existing computer simulation techniques. And based on the principle of directed evolution, the accumulated dominant traits are the most suitable for the added screening conditions, so the mutants obtained must also be the best individuals among all mutants.
  • the activity of the mutant DNA polymerase provided by the present invention is at least 1.2 times that of wild-type DNA polymerase (SEQ ID NO.: 2); preferably at least 1.3 times; more preferably The ground is at least 1.5 times, such as 2 times or more.
  • the activity test methods of the mutant DNA polymerase and the wild-type DNA polymerase are as follows:
  • the PCR product was purified by ethanol precipitation, the light absorption value of the product at 260nm was measured, and the total amount (ng) of the PCR product corresponding to each cycle number was calculated.
  • primer pairs are used in the PCR reaction:
  • pET28_F primer TACGGTTAACCCTTTGAATCA (SEQ ID NO.: 9)
  • pET28_R primer GTTACCTGGTTAAACTGTACT (SEQ ID NO.: 10).
  • Dividing the total amount of PCR products obtained using the Taq enzyme mutant by the total amount of PCR products obtained using the wild-type Taq enzyme is the multiple of the activity of the Taq enzyme mutant compared to the wild-type Taq enzyme.
  • a person of ordinary skill in the art can use conventional methods to obtain the Taq enzyme gene sequence of the present invention, such as full artificial synthesis or PCR synthesis.
  • a preferred synthesis method is the asymmetric PCR method.
  • the asymmetric PCR method uses a pair of primers of unequal amounts to produce a large amount of single-stranded DNA (ssDNA) after PCR amplification. This pair of primers are called non-restricted primers and restricted primers, and the ratio is generally 50-100:1.
  • the amplified products are mainly double-stranded DNA, but when the restricted primers (low-concentration primers) are consumed, the PCR guided by the non-restrictive primers (high-concentration primers) will be A large amount of single-stranded DNA is produced.
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the Taq enzyme of the present invention can be expressed or produced by conventional recombinant DNA technology, including the steps:
  • a method well known to those skilled in the art can be used to construct an expression vector containing the coding DNA sequence of the Taq enzyme of the present invention and suitable transcription/translation control signals, preferably a commercially available vector: pET28. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the recombinant vector includes a promoter, a target gene and a terminator in the 5'to 3'direction. If necessary, the recombinant vector may also include the following elements: protein purification tag; 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; selection marker (antibiotic resistance gene, fluorescent protein, etc.) ; Enhancer; or operator.
  • the methods for preparing recombinant vectors are well known to those of ordinary skill in the art.
  • the expression vector can be a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors. In short, as long as it can replicate and stabilize in the host, any plasmid and vector can be used.
  • Those of ordinary skill in the art can use well-known methods to construct a vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the expression vector of the present invention can be used to transform an appropriate host cell so that the host can transcribe the target RNA or express the target protein.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells .
  • prokaryotic cell such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells .
  • the host When the host is a prokaryotic organism (such as Escherichia coli), it can be treated with the CaCl 2 method or electroporation method.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc. The transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain transgenic plants.
  • operably linked means that the target gene to be transcribed and expressed is linked to its control sequence in a conventional manner in the art to be expressed.
  • the engineered cell can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media, and the culture is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • DO dissolved oxygen
  • the types of feeding should include carbon sources such as glycerol, methanol, glucose, etc., which can be fed separately or mixed;
  • any conventional induction concentration can be used in the present invention, and the IPTG concentration is usually controlled at 0.1-1.5 mM;
  • the target protein Taq enzyme of the present invention is stored in E. coli cells, the host cells are collected by a centrifuge, and then the host cells are broken by high pressure, mechanical force, enzymatic cell lysis or other cell disruption methods, and the recombinant protein is released.
  • the high pressure method is preferred.
  • the host cell lysate can be preliminarily purified by methods such as flocculation, salting out, ultrafiltration, etc., and then purified by chromatography, ultrafiltration, etc., or it can be directly purified by chromatography.
  • Chromatography techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography and other techniques. Commonly used chromatography methods include:
  • Anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is high, which affects the combination with the ion exchange medium, it is necessary to reduce the salt concentration before performing ion exchange chromatography.
  • the sample can be replaced by dilution, ultrafiltration, dialysis, gel filtration chromatography and other means to balance the buffer solution until it is similar to the corresponding ion exchange column balance solution system, and then load the sample for salt concentration or pH gradient elution.
  • Hydrophobic chromatography media include (but are not limited to): Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose.
  • the sample is increased in salt concentration by adding NaCl, (NH 4 ) 2 SO 4, etc., and then loaded, and eluted by reducing the salt concentration.
  • Hydrophobic chromatography is used to remove contaminated proteins with large differences in hydrophobicity.
  • Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, Sephadex. Replace the buffer system by gel filtration chromatography, or further refine it.
  • Affinity chromatography media include (but are not limited to): HiTrap TM Heparin HPColumns.
  • Ultrafiltration media include: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Purification and concentration can be achieved through membrane filtration.
  • the heat-resistant DNA polymerase mutant with high amplification activity of the present invention has a significantly higher amount of product amplified under the same number of PCR cycles than the wild-type Taq enzyme.
  • the heat-resistant DNA polymerase mutant with high amplification activity of the present invention requires a significantly shorter time than wild-type Taq enzyme to amplify and produce the same amount of product under the same conditions, and therefore can significantly improve detection. efficient.
  • T1-423_PF 5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.: 4)
  • T1-423_PR 5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.: 5)
  • the PCR product was purified and recovered with a DNA gel recovery kit, digested with NdeI and XhoI, and ligated to the pET28a vector. Sequencing confirmed that the sequence was correct.
  • the resulting plasmid was named Taq(1-423)-pET28
  • TMu_F 5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.: 7)
  • TMu_R 5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.: 8)
  • the PCR product was digested with BsmBI and XhoI, and then ligated with the Taq(1-423)-pET28 plasmid digested with BsmBI and XhoI.
  • the ligation product was transformed into BL21(DE3) expression host bacteria, and the number of transformants was counted.
  • the Taq enzyme mutant plasmid was transformed into BL21(DE3) expression strain, and the Taq enzyme mutant library was induced to express.
  • the BL21 (DE3) induced expression bacteria containing the Taq enzyme mutation library were dispersed and packaged with an emulsion PCR system, and the PCR reaction was performed to amplify the DNA containing the Taq enzyme mutation fragment. Then, the DNA fragment amplified by emulsion PCR was amplified by high-fidelity PCR with Taq enzyme-specific primers, and the amplified DNA product was re-cloned into the pET28a expression vector to complete a screening process.
  • IPTG isopropylthiogalactoside
  • pET28_F primer TACGGTTAACCCTTTGAATCA (SEQ ID NO.: 9)
  • pET28_R primer GTTACCTGGTTAAACTGTACT (SEQ ID NO.: 10)
  • step 4 Use the product of step 4) as a template to perform PCR secondary amplification
  • the PCR program is as follows: 95°C for 5 minutes, 20 cycles X (95°C for 30 seconds, 62°C for 30 seconds, 72°C for 2 minutes) 72°C for 5 minutes, 4°C ⁇
  • Taq_F primer ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG (SEQ ID NO.: 11)
  • Taq_R primer TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC (SEQ ID NO.: 12)
  • the PCR product is purified with a DNA product purification recovery kit, and then re-attached to the pET28a expression vector. So far complete a round of screening
  • the second round of screening 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes) X 25 cycles, 72°C for 5 minutes, 4°C ⁇
  • the third round of screening 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute) X 20 cycles, 72°C for 5 minutes, 4°C ⁇
  • the fourth round of screening 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds) X 15 cycles, 72°C for 5 minutes, 4°C ⁇
  • the DNA sequence of the Taq enzyme mutant selected in Example 3 was sequenced to determine the mutation of its amino acid sequence, and the high-frequency mutation sites and their mutation forms were counted.
  • the 20 mutants with good amplification activity were sequenced, and the statistics of their amino acid mutations are shown in the above table. It can be seen: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, H785 recurred frequently in 20 mutants, proving that its mutation has a significant impact on the amplification activity of Taq enzyme.
  • PCR program 95°C for 5 minutes, n cycles X (95°C for 15 seconds, 55°C for 15 seconds, 72°C for 10 seconds), 4C ⁇
  • the amount of products amplified by Taq enzyme mutants 1 to 20 under the same number of PCR cycles is significantly higher than that of wild-type Taq enzyme.
  • the amount of product obtained when mutant 1 was amplified for 20 cycles was equivalent to the amount of product obtained by 30 cycles of wild-type Taq enzyme amplification; under the same conditions of 30 amplification cycles, the amount of product obtained by mutant 1 was Reached more than 2.5 times the amount of wild-type Taq enzyme product.
  • NC (ORF1ab/N) PCR reaction solution A NC (ORF1ab/N) positive quality control product nucleic acid extracts are all made of 2019 new coronavirus (2019-nCoV) ORF1ab N nucleic acid detection kit (PCR-fluorescent probe method) (Sun Yat-Sen University Daan Gene Co., Ltd.) Provided, MMLV reverse transcriptase and RNase Inhibitor were prepared by Sun Yat-Sen University Daan Gene Co., Ltd.
  • the PCR program settings are as follows:
  • NC (ORF1ab/N) positive quality control products with different concentration gradients of Taq enzyme mutants and wild-type Taq enzymes are as follows:

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Abstract

一种具有高扩增活性的耐热DNA聚合酶突变体，应用蛋白质定向进化技术，对针对Taq酶的聚合酶活性结构域构建随机突变库，通过逐步添加筛选压力，使不适合的突变自然淘汰，具有优势性状的突变逐步积累，最终筛选出一系列对Taq酶扩增性能、聚合性能有关键作用的氨基酸位点及其突变，并得到高扩增性能的Taq酶突变体。

Description

一种具有高扩增活性的耐热DNA聚合酶突变体 技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种具有高扩增活性的耐热DNA聚合酶突变体。
背景技术
Taq酶是一种来源于耐热性细菌Thermus aquaticus的耐热性DNA聚合酶，分子量94KDa，在镁离子存在的条件下，其最适反应温度为75-80℃，在95℃下的活性半衰期为40分钟，具有5’-3’核酸外切酶活性。由于其具有耐高温的特性，因此广泛用于聚合酶链式反应(PCR)，是核酸扩增、检测等反应的首选用酶。商品化Taq酶使用大肠杆菌原核表达系统进行克隆及表达。现代分子生物检测技术对PCR反应的灵敏度、精确度、耐用性要求越来越高，野生型Taq酶无法完全满足实际应用的需求。为使其更加适应特定技术的使用，对Taq酶序列的突变改造进行了很多的尝试，如添加DNA结合结构域使其具有更强的延伸活性(Wang Y(2004).A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro.Nucleic Acids Res 32,1197–1207))；通过定点突变、删除结构域使其具有更高的保真性(Suzuki M,Yoshida S,Adman ET,Blank A,Loeb LA(2000)Thermus Aquaticus DNA polymerase I mutants with altered fidelity.Interacting mutations in the O-Helix.J Biol Chem 275:32728–32735)、更高的DNA聚合活性(Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.Front Microbiol 5:461.doi:10.3389/fmicb.2014.00461)、耐受高浓度抑制剂(Zhang Z,Kermekchiev MB,Barnes WM(2010)Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.J Mol Diagn 12:152–161)、降低5’-3’核酸外切酶活性(Vainshtein I,Atrazhev A,Eom SH,Elliott JF,Wishart DS,Malcolm BA(1996)Peptide rescue of an N-Terminal truncation of the Stoffel fragment of Taq DNA polymerase.Protein Sci 5:51785–51792)。
对Taq酶的改造主要有以下几种途径。1：增加结构域，使其具有新的性能。 如加入单链结合结构域(SSB)或DNA结合蛋白Sso7，增强Taq酶对引物及模板DNA的结合能力，使其具有更强的延伸能力和持续合成能力，适合用于长片段DNA的扩增反应。但增加结构域会直接增大Taq酶的分子量，有可能使Taq酶的溶解性和稳定性降低。降低原核表达生产的产量。2：去除Taq酶上非必要的结构域。如将5’-3’核酸外切酶结构域(Taq酶N端前280个氨基酸)删除，使Taq酶只保留核酸聚合酶活性区域，降低高浓度Taq酶对引物及模板DNA造成降解的可能性，达到提高Taq酶聚合活性的目的。但此方法得到的Taq酶突变体没有了5’-3’核酸外切酶活性，不适合用于基于Taq man探针法的定量PCR反应，可使用范围有限。3：定点突变法。对活性位点、镁离子结合位点、DNA结合位点的氨基酸进行定点突变，提高各位点对底物、模板及引物的亲和力，从而提高对各种抑制剂的耐受性。由于蛋白质结构的复杂性，一些远离活性位点的氨基酸也有可能对酶的整体结构产生影响，因此只对特定几个活性中心位点的氨基酸进行突变，难以从整体上对酶进行改造。而且现有的计算机模拟技术难以预测各个位点突变对整体结构会产生什么影响，用定点突变法制备突变体以及进行突变体筛选的工作量非常大，而且效率低下，一些可能对活性有重大影响的位点无法被识别。
发明内容
本发明的目的在于提供一种具有高扩增活性的耐热DNA聚合酶突变体。
在本发明的第一方面，提供了一种突变的DNA聚合酶，所述突变的DNA聚合酶在选自下组的一个或多个位点发生突变：V453、F495、E507、K508、T509、A518、S624、Y672、E734、R737、F749、T757、L764、H785，其中，氨基酸残基编号采用SEQ ID NO.2所示的编号。
在另一优选例中，所述突变的DNA聚合酶的活性是野生型DNA聚合酶(SEQ ID NO.:2)的至少1.5倍；优选地至少2倍；更优选地至少3倍。
在另一优选例中，所述野生型DNA聚合酶的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述突变的DNA聚合酶的氨基选序列与SEQ ID NO.2相比具有至少80％的同源性；更优选地，具有至少90％的同源性；最优选地，具有至少95％的同源性；如具有至少96％、97％、98％、99％的同源性。
在另一优选例中，所述突变的DNA聚合酶选自下组的突变体1-20：
Figure PCTCN2020088341-appb-000001
Figure PCTCN2020088341-appb-000002
在另一优选例中，所述突变的DNA聚合酶中突变位点的数量为1-4个，优选为2或3个。
在另一优选例中，所述突变的DNA聚合酶选自表2中的各具体突变体酶。
在另一优选例中，所述突变的DNA聚合酶包括表2中的各具体突变体酶的突变位点。
在另一优选例中，所述突变的DNA聚合酶在SEQ ID NO.:2所示的野生型DNA聚合酶基础上进行突变，并且所述突变的DNA聚合酶包括选自下组的突变位点：
(1)E507A、K508L、E734E、F749K；
(2)K508L、V453A、R737K
(3)E734G
(4)F749G、K508L、L764K
(5)E507Q、T757S
(6)H785G
(7)S624T、F749V
(8)E734F、F749V
(9)K508L、R737W、Y672R
(10)E507H、H785L
(11)A518Q、E734M
(12)F495R、F749T
(13)K508L、F749T、E734F
(14)R737P、S624K
(15)T757W、V453G、E507M
(16)F749E、H785G、F495G
(17)E734F、Y672P
(18)T509L、H785K
(19)E734G、T757S、L764Q；和
(20)K508L、V453A、A518Q。
本发明的第二方面，提供了一种多核苷酸分子，所述多核苷酸分子编码本发明第一方面所述的突变的DNA聚合酶。
本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的核酸分子。
本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第一方面所述的载体或染色体整合有本发明第二方面所述的核酸分子。
在另一优选例中，所述宿主细胞为原核细胞、或真核细胞。
在另一优选例中，所述原核细胞为大肠杆菌。
在另一优选例中，所述真核细胞为酵母细胞。
本发明的第五方面，提供了一种制备本发明第一方面所述的突变的DNA聚合酶的方法，包括步骤：
(i)在适合的条件下，培养本发明第四方面所述的宿主细胞，从而表达出所述的突变的DNA聚合酶；和
(ii)分离所述的突变的DNA聚合酶。
在另一优选例中，所述步骤(i)中培养所述宿主细胞的温度为20℃-40℃；优选为25℃-37℃，如35℃。
本发明的第六方面，提供了一种试剂盒，所述试剂盒包含本发明第一方面所述的突变的DNA聚合酶。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人通过广泛而深入的研究，应用蛋白质定向进化技术，对针对Taq酶的聚合酶活性结构域构建随机突变库，通过逐步添加筛选压力，使不适合的突变自然淘汰，具有优势性状的突变逐步积累，最终筛选出一系列对Taq酶扩增性能、聚合性能有关键作用的氨基酸位点及其突变，并得到高扩增性能的Taq酶突变体。在此基础上，完成了本发明。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
Taq酶
Taq酶广泛用于聚合酶链式反应(PCR)，是核酸扩增、检测等反应的首选用酶。商品化Taq酶使用大肠杆菌原核表达系统进行克隆及表达。
野生型Taq酶DNA序列如下：
Figure PCTCN2020088341-appb-000003
野生型Taq酶氨基酸序列如下：
Figure PCTCN2020088341-appb-000004
Figure PCTCN2020088341-appb-000005
本发明通过定向进化的方式，筛选出与Taq酶扩增活性高度相关的氨基酸位点及其突变方式。相关突变氨基酸位点包括：V453、F495、E507、K508、T509、A518、S624、Y672、E734、R737、F749、T757、L764、和H785，氨基酸残基编号依据SEQ ID NO.:2。将上述氨基酸位点突变成任意的其他氨基酸可以获得更高活性的Taq酶突变体，优选的突变形式包括：E507A/Q/H/M、K508L、E734G/F/M、F749K/G/V/T/E、L764K/Q、V453A/G、R737K/W/P、T757S/W、H785G/L/K、S624T/K、Y672R/P、A518Q、F495G/R、T509L。
本发明通过定向进化技术，从随机突变库中筛选出对Taq酶活性具有高度相关性的氨基酸位点及其突变方式，突变体数量是定点突变的10 5倍，更有利于筛选出具有协同效应的突变位点，而这些位点是无法通过现有的计算机模拟技术预测出来的。而且基于定向进化的原理，积累的优势性状是最适应所添加的筛选条件的，因此得到的突变体也肯定是所有突变体中的最优个体。
在本发明的一个优选地实施方式中，本发明所提供的突变的DNA聚合酶的活性是野生型DNA聚合酶(SEQ ID NO.:2)的至少1.2倍；优选地至少1.3倍；更优选地至少1.5倍，如可以为2倍以上。
在本发明的一个优选地实施方式中，突变的DNA聚合酶和野生型DNA聚合酶(SEQ ID NO.:2)的活性测试方法如下：
Figure PCTCN2020088341-appb-000006
Figure PCTCN2020088341-appb-000007
PCR程序：95℃ 5分钟，30次循环(95℃ 15秒、55℃ 15秒、72℃ 10秒)、4C∞
乙醇沉淀纯化PCR产物，测定产物在260nm处的光吸收值，计算各循环次数所对应的PCR产物的总量(ng)。
其中，PCR反应中使用如下引物对：
pET28_F引物：TACGGTTAACCCTTTGAATCA(SEQ ID NO.:9)
pET28_R引物：GTTACCTGGTTAAACTGTACT(SEQ ID NO.:10)。
将使用Taq酶突变体获得的PCR产物的总量除以使用野生型Taq酶获得的PCR产物的总量即为Taq酶突变体相较于野生型Taq酶的活性倍数。
本领域的普通技术人员可以使用的常规方法获得本发明的Taq酶基因序列，例如全人工合成或PCR法合成。一种优选的合成法为不对称PCR法。不对称PCR法是用不等量的一对引物，PCR扩增后产生大量的单链DNA(ssDNA)。这对引物分别称为非限制引物与限制性引物，其比例一般为50-100∶1。在PCR反应的最初10-15个循环中，其扩增产物主要是双链DNA，但当限制性引物(低浓度引物)消耗完后，非限制性引物(高浓度引物)引导的PCR就会产生大量的单链DNA。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明的Taq酶可以通过常规的重组DNA技术进行表达或生产，包括步骤：
(1)用编码本发明蛋白的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化目的蛋白质，从而获得Taq酶。
本领域的技术人员熟知的方法能用于构建含本发明Taq酶的编码DNA序列和合适的转录/翻译控制信号的表达载体，优选市售的载体：pET28。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选包含一个或 多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
所述重组载体在5'到3'方向上包括：启动子，目的基因和终止子。如果需要，所述重组载体还可以包括以下元件：蛋白纯化标签；3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；选择标记(抗生素抗性基因、荧光蛋白等)；增强子；或操作子。
用于制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要其能够在宿主体内复制和稳定，任何质粒和载体都可以被采用。
本领域普通技术人员可以采用熟知的方法构建含有本发明启动子和/或目的基因序列的载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明的表达载体，可以用于转化适当的宿主细胞，以使宿主转录目的RNA或表达目的蛋白质。宿主细胞可以是原核细胞，如大肠杆菌、谷氨酸棒杆菌、黄色短杆菌、链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
术语“可操作连接”是指将准备转录表达的目的基因以一种本领域的常规方式连接到它的控制序列以被表达。
工程菌的培养和目的蛋白发酵生产
在获得工程细胞后，便可在适合的条件下培养工程细胞，表达本发明的基因序列所编码的蛋白。根据宿主细胞的不同，培养中所用的培养基可选自各种常规培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在本发明中，可采用常规的发酵条件。代表性的条件包括(但并不限于)：
(a)就温度而言，Taq酶的发酵及诱导温度保持在25-37℃；
(b)就诱导期的pH值而言，诱导期pH控制在3-9；
(c)就溶氧(DO)而言，DO控制在10-90％，溶氧的维持可以用氧气/空气混合气体的通入来解决；
(d)就补料而言，补料种类宜包括甘油、甲醇、葡萄糖等碳源，可单独补料或混合补料；
(e)就诱导期IPTG浓度而言，常规诱导浓度都可用于本发明，通常IPTG浓度控制在0.1-1.5mM；
(f)就诱导时间而言，没有特别限制，通常为2-20小时，较佳地为5-15小时。
本发明的目的蛋白Taq酶存在大肠杆菌细胞胞内，通过离心机收集宿主细胞，然后通过高压、机器力、酶解细胞被或其他细胞破碎方法破碎宿主细胞，释放重组蛋白，优选的是高压法。宿主细胞裂解液可通过絮凝、盐析、超滤等方法进行初步纯化后再进行层析、超滤等纯化，也可直接进行层析纯化。
层析技术包括阳离子交换层析、阴离子交换层析、凝胶过滤层析、疏水层析、亲和层析等技术。常用的层析方法包括：
1.阴离子交换层析：
阴离子交换层析介质包括(但不限于)：Q-Sepharose、DEAE-Sepharose。如果发酵样品的盐浓度较高，影响与离子交换介质的结合，则在进行离子交换层析前需降低盐浓度。样品可以用稀释、超滤、透析、凝胶过滤层析等手段进行平衡缓冲液的更换，直至与对应的离子交换柱平衡液系统相似，然后上样，进行盐浓度或pH的梯度洗脱。
2.疏水层析：
疏水层析介质包括(但不限于)：Phenyl-Sepharose、Butyl-Sepharose、Octyle-Sepharose。样品通过添加NaCl、(NH 4) 2SO 4等方式提高盐浓度，然后上样，通过降低盐浓度方法洗脱。通过疏水层析除去疏水性有较大差异的杂蛋白。
3.凝胶过滤层析
疏水层析介质包括(但不限于)：Sephacryl、Superdex、Sephadex类。通过凝胶过滤层析更换缓冲体系，或进一步精纯。
4.亲和层析
亲和层析介质包括(但不限于)：HiTrap TMHeparinHPColumns。
5.膜过滤
超滤介质包括：有机膜如聚砜膜、无机膜如陶瓷膜、金属膜类。通过膜过滤可以达到纯化和浓缩的目的。
本发明的主要优点在于：
(1)本发明的具有高扩增活性的耐热DNA聚合酶突变体在同等PCR循环次数下扩增得到的产物量比野生型Taq酶有显著的提高。
(2)本发明的具有高扩增活性的耐热DNA聚合酶突变体在同等条件下扩增产生同等量的产物所需的时间比野生型Taq酶有显著的缩短，因此，能够显著提高检测效率。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1：Taq酶随机突变质粒的构建
用低保真度PCR(Error-PCR)扩增Taq酶的聚合酶活性结构域DNA序列(423－831位氨基酸编码序列)，突变发生率为0.3％，然后与Taq酶的其余编码序列(1－423位氨基酸序列)连接，克隆至pET28a原核表达载体中，得到Taq酶随机突变质粒。具体步骤如下：
1)以Taq-pET28a质粒为模板，设计引物T(1－423)扩增Taq(1－423)片段。
Taq(1-423)DNA Seq
Figure PCTCN2020088341-appb-000008
Figure PCTCN2020088341-appb-000009
T1-423_PF：5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.:4)
T1-423_PR：5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.:5)
PCR反应体系及程序：
Figure PCTCN2020088341-appb-000010
PCR程序：95℃ 3分钟，(95℃ 30秒，60℃ 30秒，72℃ 1分钟)×25个循环，72℃ 3分钟，4℃保存
PCR产物用DNA胶回收试剂盒纯化回收，用NdeI及XhoI酶切，连接到pET28a载体，测序确认序列正确，得到的质粒命名为Taq(1-423)-pET28
2)以Taq-pET28a质粒为模板，使用Clontech
Figure PCTCN2020088341-appb-000011
PCR Random Mutagenesis Kit(大连宝生物PT3393-2)，设计引物(TMu_F/R)扩增Taq(423-822)片段
Taq(423-832)DNA Seq
Figure PCTCN2020088341-appb-000012
Figure PCTCN2020088341-appb-000013
TMu_F：5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.:7)
TMu_R：5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.:8)
PCR反应体系及程序：
Figure PCTCN2020088341-appb-000014
95℃ 3分钟,(95℃ 30秒,60℃ 30秒，68℃ 2分钟)X25次循环，68℃ 5分钟，4℃保存
PCR产物用BsmBI及XhoI酶切，然后连接经BsmBI及XhoI酶切的Taq(1-423)-pET28质粒，连接产物转化BL21(DE3)表达宿主菌，统计转化子数量。
实施例2：Taq酶突变体的表达及定向进化筛选
Taq酶突变质粒转化BL21(DE3)表达菌株，诱导表达出Taq酶突变库。将含有Taq酶突变库的BL21(DE3)诱导表达菌用乳液PCR体系分散包裹，进行PCR反应，扩增出含有Taq酶突变片段的DNA。然后把乳液PCR扩增出来的DNA片段用Taq酶特异引物进行高保真PCR二次扩增，将扩增的DNA产物重新克隆至pET28a表达载体中，完成一次筛选过程。之后重复进行乳液PCR-二次高保真PCR-克隆至pET28a表达载体的筛选过程，同时逐步缩短每次筛选中乳液PCR的延伸时间，使具有高延伸活性和高扩增活性的突变体群体得到累积。具体步 骤如下：
1)取实施例1转化得到的转化子，接种至LB培养基中，37℃震荡培养6小时，加入终浓度0.1mM的异丙基硫代半乳糖苷(IPTG)，37℃诱导培养3小时。离心收集菌体，用ddH 2O洗涤菌体两次，最后用ddH 2O重悬菌体，测定菌体溶液在600nm处的光吸收值(OD600值)，用ddH 2O将终浓度稀释至OD600＝1.0
2)配制油相溶液
Tween-80        200ul
Triton X-100    25ul
Mineral oil     10ml
以上3种试剂合并，混合均匀
3)水相反应液的配制
将步骤1)配制好的OD600＝1.0的菌体重悬液，用ddH 2O稀释100倍，配制以下反应液
Figure PCTCN2020088341-appb-000015
pET28_F引物：TACGGTTAACCCTTTGAATCA(SEQ ID NO.:9)
pET28_R引物：GTTACCTGGTTAAACTGTACT(SEQ ID NO.:10)
4)乳液体系的制备
取200ul水相+400ul油相在2ml管中混合，在漩涡振荡器上高速振荡10分钟，取5个PCR管，每个分装100ul混合液，P℃R程序：95℃ 5分钟，(95℃ 30秒、55℃ 30秒、72℃ 2分钟)X 25次循环、72℃ 5分钟，4℃∞
5)将乳液PCR产物转移至1.5ml管，加入166ul水饱和乙醚，漩涡振荡30秒，12000rpm离心10分钟，转移走下层液相，室温静置10分钟待乙醚挥发，采用酚氯仿法抽提纯化液相产物，然后乙醇沉淀过夜回收产物。
6)高保真PCR二次扩增产物
以步骤4)产物为模板，进行PCR二次扩增
Figure PCTCN2020088341-appb-000016
PCR程序如下：95℃ 5分钟，20次循环X(95℃ 30秒、62℃ 30秒、72℃ 2分钟)72℃ 5分钟、4℃∞
Taq_F引物：ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG(SEQ ID NO.:11)
Taq_R引物：TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC(SEQ ID NO.:12)
7)PCR产物用DNA产物纯化回收试剂盒纯化，然后重新连接pET28a表达载体。至此完成一轮筛选
8)将重新连接pET28a载体的转化子重复步骤(1)-(6)，按下表的程序改变乳液PCR的条件，对突变库逐步添加选择压力
第二轮筛选：95℃ 5分钟，(95℃ 30秒、55℃ 30秒、72℃ 1.5分钟)X 25次循环、72℃ 5分钟，4℃∞
第三轮筛选：95℃ 5分钟，(95℃ 30秒、55℃ 30秒、72℃ 1分钟)X 20次循环、72℃ 5分钟，4℃∞
第四轮筛选：95℃ 5分钟，(95℃ 30秒、55℃ 30秒、72℃ 30秒)X 15次循环、72℃ 5分钟，4℃∞
经4轮筛选后，所得的Taq酶突变体转化子进入实施例3的高通量筛选
实施例3：Taq酶突变体的高通量筛选
从实施例2得到的突变库中随机挑取384个单克隆，经培养及诱导表达后，用高通量PCR反应测试其扩增活性，从中挑选出20个具有高扩增活性的突变体。具体步骤如下：
1)挑取384个单克隆，接种到LB培养基中，37℃培养6小时，加入终浓度0.1mM的异丙基硫代半乳糖苷(IPTG)，37℃诱导培养3小时
2)离心收集诱导培养后菌体，加入含0.1mg/ml溶菌酶的裂解液(50Mm Tris，50Mm NaCl，5％甘油 pH8.5)，重悬菌体，37℃孵育10分钟，75℃加热30分钟。然后12000rpm离心10分钟，取上清液。
3)取96孔PCR板，每孔加入以下反应组分
Figure PCTCN2020088341-appb-000017
PCR程序：95℃ 5分钟，20次循环X(95℃ 30秒、62℃ 30秒、72℃ 60秒)、4C∞
PCR产物取5ul进行琼脂糖凝胶电泳，对比各单克隆制备的上清液的PCR产物的产量，选出产量最高20个单克隆。各突变体的扩增产量是野生型扩增产量的1.2倍制2倍。
实施例4：优势Taq酶突变体突变位点的确认
对实施例3选出的Taq酶突变体进行的DNA序列测序，确定其氨基酸序列的突变情况，统计高频突变位点及其突变的形式。
表1
Figure PCTCN2020088341-appb-000018
Figure PCTCN2020088341-appb-000019
对扩增活性较好的20个突变体进行测序，统计其氨基酸突变情况如上表，可见：V453、F495、E507、K508、T509、A518、S624、Y672、E734、R737、F749、T757、L764、H785在20个突变体高频重复出现，证明其突变对Taq酶的扩增活性有显著的影响。
实施例5经突变改造后的Taq酶与野生型Taq酶的对比
取Taq酶突变体，经表达纯化后，与野生型Taq酶进行以下扩增能力测试：
Figure PCTCN2020088341-appb-000020
PCR程序：95℃ 5分钟，n次循环X(95℃ 15秒、55℃ 15秒、72℃ 10秒)、4C∞
配制以上反应液，PCR扩增15、20、25、30个循环，乙醇沉淀纯化PCR产物，测定产物在260nm处的光吸收值，计算各循环次数所对应的PCR产物的总量(ng)。结果如下：
表2
Figure PCTCN2020088341-appb-000021
Figure PCTCN2020088341-appb-000022
从以上结果可见，Taq酶突变体1至20在同等PCR循环次数下扩增得到的产物量比野生型Taq酶有显著的提高。其中，突变体1扩增20个循环时得到 的产物量已经跟野生型Taq酶扩增30个循环所得的产物量相当；在同为30个扩增循环的条件下，突变体1获得产物量达到了野生型Taq酶产物量的2.5倍多。
实施例6经突变改造后的Taq酶在新型冠状病毒SARS-CoV-2荧光定量PCR检测试剂盒上的应用
挑选Taq酶突变体1#、6#、17#，按下表配制反应体系
Figure PCTCN2020088341-appb-000023
其中NC(ORF1ab/N)PCR反应液A，NC(ORF1ab/N)阳性质控品核酸提取物均由2019新型冠状病毒(2019-nCoV)ORF1ab N核酸检测试剂盒(PCR-荧光探针法)(中山大学达安基因股份有限公司)提供，MMLV逆转录酶、RNase Inhibitor均由中山大学达安基因股份有限公司制备。PCR程序设置如下：
Figure PCTCN2020088341-appb-000024
各Taq酶突变体及野生型Taq酶扩增不同浓度梯度的NC(ORF1ab/N)阳性质控品ct值如下：
Figure PCTCN2020088341-appb-000025
从以上结果可见，Taq酶突变体在SARS-CoV-2荧光定量PCR检测试剂盒上的性能比野生型Taq酶有明显的提高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种突变的DNA聚合酶，其特征在于，所述突变的DNA聚合酶在选自下组的一个或多个位点发生突变：V453、F495、E507、K508、T509、A518、S624、Y672、E734、R737、F749、T757、L764、H785，其中，氨基酸残基编号采用SEQ ID NO.2所示的编号。
  2. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶的活性是野生型DNA聚合酶的至少1.2倍。
  3. 如权利要求2所述的DNA聚合酶，其特征在于，所述野生型DNA聚合酶的氨基酸序列如SEQ ID NO.:2所示。
  4. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶中突变位点的数量为1-4个。
  5. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶的活性是野生型DNA聚合酶的至少1.5倍。
  6. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶中突变位点的数量为2或3个。
  7. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶的氨基选序列与SEQ ID NO.2相比具有至少90％的同源性。
  8. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶的氨基选序列与SEQ ID NO.2相比具有至少98％的同源性。
  9. 如权利要求1所述的DNA聚合酶，其特征在于，所述突变的DNA聚合酶在SEQ ID NO.:2所示的野生型DNA聚合酶基础上进行突变，并且所述突变的DNA聚合酶包括选自下组的突变位点：
    (1)E507A、K508L、E734E、F749K；
    (2)K508L、V453A、R737K；
    (3)E734G；
    (4)F749G、K508L、L764K；
    (5)E507Q、T757S；
    (6)H785G；
    (7)S624T、F749V；
    (8)E734F、F749V；
    (9)K508L、R737W、Y672R；
    (10)E507H、H785L；
    (11)A518Q、E734M；
    (12)F495R、F749T；
    (13)K508L、F749T、E734F；
    (14)R737P、S624K；
    (15)T757W、V453G、E507M；
    (16)F749E、H785G、F495G；
    (17)E734F、Y672P；
    (18)T509L、H785K；
    (19)E734G、T757S、L764Q；和
    (20)K508L、V453A、A518Q。
  10. 一种多核苷酸分子，其特征在于，所述多核苷酸分子编码权利要求1所述的突变的DNA聚合酶。
  11. 一种载体，其特征在于，所述载体含有权利要求10所述的核酸分子。
  12. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求11所述的载体或染色体整合有权利要求10所述的核酸分子。
  13. 一种制备权利要求1所述的突变的DNA聚合酶的方法，其特征在于，包括步骤：
    (i)在适合的条件下，培养权利要求12的宿主细胞，从而表达出所述的突变的DNA聚合酶；和
    (ii)分离所述的突变的DNA聚合酶。
  14. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1所述的突变的DNA聚合酶。
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WO2024000364A1 (zh) * 2022-06-30 2024-01-04 江南大学 一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用

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